1) Adenylate cyclase in mammalian cells is regulated by hormones and drugs that exert both positive (Beta-adrenergic agonists) and negative (opiates, muscarinic, cholinergic) effects. A protein conduct secreted by Bordetella pertussis, termed Pertussis toxin, blocks the action of inhibitory agents by catalyzing the ADP-ribosylation of a regulatory component (Gi) of cyclase. In the absence of cellular products, the toxin hydrolyzes NAD to ADP-ribose and nicotinamide; the reaction is enhanced by thiols. Toxin-catalyzed ADP-ribosylation of Gi in some membrane preparations (e.g., human erythrocytes) was dependent on thiol. In other membrane systems (NG108-15 hybrid cells), ADP-ribosylation was enhanced by thiol but not dependent on these agents. 2) Thiol:protein oxidoreductase accelerates the reduction of proteins by thiols; it may be involved in the intracellular activation of choleragen (cholera toxin) and pertussis toxin. Enzyme activity was demonstrated in cultured human fibroblasts; activity was dependent on growth conditions. Increased activity was observed in the presence of insulin and fetal calf serum. 3) Animal cells contain enzymes that catalyze mono-ADP-ribosylation reactions similar to those catalyzed by the bacterial toxins. Erythrocytes have two soluble NAD:arginine ADP-ribosyltransferases; these enzymes have different regulatory, kinetic and physical properties. The purified NAD:arginine ADP-ribosyltransferases were used to synthesize defined ADP-ribose-agmatine or ADP-riboseprotein linkages. The ribosyl-acceptor bond formed by both transferases with either agmatine or protein as acceptor appeared to be identical based on general stability properties and, in particular, its sensitivity to digestion with hydroxylamine. The bond was considerably more stable than that synthesized by the poly(ADP-ribose) synthetase in which the carboxyl moiety of glutamate serves as the ADP-ribose acceptor. It thus may be possible to classify ADP-ribose linkages in vivo based on the known reactivities to hydroxylamine of the ADP-ribose-protein bonds synthesized by the transferases or the synthetase.